We have examined the effect of illumination upon the patterns of protein synthesis in the filamentous ascomycete Neurospora crassa by pulse labelling and two-dimensional gel electrophoresis. Light did not affect overall rates of protein synthesis but did induce the synthesis of six novel polypeptides whose appearance followed a temporally regulated pattern. When translation products of mRNA from illuminated cultures and dark control cultures were compared it was found that the synthesis of five out of six of the polypeptides specific to illuminated cultures could be seen in vitro. We believe that this is consistent with the hypothesis that light regulates the transcription of some genes in N. crassa, although we cannot exclude effects on mRNA stability or the control of precursor splicing.
We have examined methods for broadening and stabilising pH gradients used in the first dimension of two-dimensional gel electrophoresis. The replacement of typical strong electrolytes with weak electrolytes as reservoir anolyte and catholyte allows the generation of broad (3.5 pH unit) gradients that are stable for at least 28 000 volt x hours (Vh). Protein patterns form within 10 000 Vh are stable throughout the subsequent focusing. Large quantities ofprotein, up to 6 mg, can be loaded onto such gels by mixing samples into the gel prior to polymerisation. We found no evidence for extensive modification of proteins because of exposure to the polymerisation reaction. The pH gradient formed in strong electrolytes in the typical O'Farrell gel system (J. Bid. Chem. 1975,250,4007-4021) could also be broadened ifthe samples were again mixed into the gel before polymerisation. This last effect appears to be due to an effect of the sample buffer used upon end loading of samples. J. A. A. Chambers et al.
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